The present invention relates to methods of treating diseases using anti-antimicrobial peptide (AMP) and/or AMP-like molecule (AML) and in particular cathelicidin type AMPs, and to methods of identifying compounds capable of regulating, decreasing or increasing activities/levels of AMPs/AMLs so as to enable treatment of diseases. More particularly, the present invention relates to methods of treating diseases by using cathelicidin or cathelicidin fragments or cathelicidin analogs or compounds capable of regulating the levels/activity of cathelicidin, such diseases including dysregulated cell proliferation/differentiation leading to bone loss or degradation, osteoporosis osteoarthritis, or to other autoimmune diseases such as multiple sclerosis, arthritis, psoriasis, and to malignancies such as carcinomas, which are associated with inflammation, to metabolic diseases, obesity, insulin resistance, diabetes type 2, diabetes type 1 and related diseases. Also, the present invention relates to methods of identifying compounds capable of regulating levels of cathelicidin or other AMPs or to increasing or to decreasing activity/levels of AMPs so as to enable treatment of diseases including autoimmune and inflammatory diseases such as, multiple sclerosis, arthritis, metabolic disorders such as diabetes, obesity and malignant diseases such as carcinomas, which are associated with inflammation, dysregulated cell proliferation/differentiation, angiogenesis and/or metastasis.
Both inhibiting endogenous cathelicidin based peptides or other AMPs as well as the use of such cathelicidin based peptides or analogs of cathelicidin peptides are effective modes of treatments for disease. As was demonstrated in WO 2004-056307 filed by the present inventors and incorporated herein, cathelicidins are immune regulators and are over expressed locally in autoimmune diseases. They are also expressed systemically through bone marrow such that normal plasma concentrations average around 1.2 ug/ml to 1.5 ug/ml (Journal of Immunological Methods 206—1997.53-59). Regulation of their expression is essential for homeostasis. AMPs are involved is skewing dendritic cell activation between Th1 and Th2 inflammatory processes via Toll-like receptors and therefore are involved in homeostasis (J Immunol. 2004 Jan. 15; 172(2):1146-56). Controlling or maintaining such homeostasis is performed by either increasing or decreasing of level/activity between the various AMPs.
Cathelicidins are mainly expressed by Vitamin D3 (calcitriol), via vitamin D3 receptor elements (VDRE) and Vitamin D3 itself has a modulating influence on cathelicidin expression both as an agonist via calcitriol/VDRE and by a negative feedback mechanisms (Marshall T BioEssays 30:173-182, 2008). This VDRE/cathelicidin pathway is unique to humans and furry/haired animals such as rodents for example whose skin is less exposed to sunlight do not possess this pathway. As shown in data included in this invention for the first time relative to prior art, cathelicidin forms a major immune regulator for diseases which are known to be also regulated by vitamin D3. These include amongst others, bone loss in Periodontitis (which is associated with low vitamin D and low cathelicidin), Obesity, Type 2 Diabetes mellitus type 1 and type 2 (which is associated with low vitamin D and Toll like receptor 4, which cathelicidin inhibits), Atherosclerosis (low vitamin D association), Hypertension (low vitamin D association), Asthma and Allergy (low vitamin D association), Osteoporosis and Ostepenia (low vitamin D association), Multiple Sclerosis (low vitamin D association), Rheumatoid arthritis (low vitamin D association), Autoimmune Diseases such as Crohn, Type 1 Diabetes (low vitamin D association), Schizophrenia (low vitamin D association), Muscle wasting disease including age associated muscle wasting (low vitamin D association as well as beta defensin overexpression), Cancer (low vitamin D association as well as Cathelicidin and beta defensin overexpression), Depression (low vitamin D association), Skin inflammation including Psoriasis (treated with vitamin D analogues), Tubeculosis and Influenza (low vitamin D association), Chronic Pain (low vitamin D association), Osteoartheritis (low vitamin D association), The Common Cold and other known diseases (The Breast Journal, Volume 14 Number 3, 2008 255-260, Photochem Photobiol. 2008 March-April; 84(2):356-65) associated with vitamin D3, commonly known as the “Sunshine vitamin” and inappropriately called a vitamin but is in fact a hormone. Data as presented in this invention indicate a common pathway of disease regulation between cathelicidin and vitamin D3. For this reason, the inventor reasons that diseases such as schizophrenia and depression which cannot be modeled suitably by animals are also regulated by cathelicidin.
Diseases, such as malignant, autoimmune, and allergic diseases, which are associated with biological processes such as inflammation, dysregulated cell proliferation/differentiation, and dysregulated cell proliferation/differentiation balance include a vast range of highly debilitating and/or lethal pathologies of great economic impact, for which no satisfactory treatment methods are presently available. For example autoimmune diseases represent diseases of major clinical and economic impact. These include major diseases such as psoriasis, rheumatoid arthritis, type I diabetes, inflammatory bowel diseases, and multiple sclerosis for which no satisfactory treatment methods are available. Similarly, malignant diseases, such as skin carcinoma, breast carcinoma, colon carcinoma, head and neck carcinoma, hepatic carcinoma, lung carcinoma, renal cell carcinoma, urinary bladder carcinoma, and the like, represent numerous lethal diseases for which no satisfactory treatment methods are available.
There is an urgent and long-felt need for optimal methods of treating such diseases which are associated with inflammation, dysregulated cell/tissue proliferation/differentiation and autoimmunity.
The epithelial lining of the skin, gastrointestinal tract and bronchial tree produces a number of peptides with antimicrobial activities termed antimicrobial peptides (AMPs), which appear to be involved in both innate host defense and adaptive immune responses (Yang D. et al., 2001. Cell Mol Life Sci. 58:978-89). AMPs are cationic peptides which display antimicrobial activity at physiological concentrations under conditions prevailing in the tissues of origin. AMP synthesis and release is regulated by microbial signals, developmental and differentiation signals, cytokines and in some cases neuroendocrine signals in a tissue-specific manner. Their mode of action is unknown, however the leading theory claims that permeabilization of target membranes is the crucial step in AMP-mediated antimicrobial activity and cytotoxicity. AMPs are classified into two major groups in humans; cathelicidins and defensins. AMPs appear to have common characteristics that enable them to affect mammalian cells in a way that does not necessarily function through a ligand-receptor pathway, and that, being small, and highly ionic or hydrophobic or structurally amphiphilic, AMPs can bind mammalian cell membranes. They are able to penetrate through the cell membrane to the cytoplasm. For example, it was shown that granulysin penetrates and damages human cell membranes dependent upon negative charge (J. Immunol., 2001, 167:350-356). At high concentrations they are cytotoxic to cells, they tear through the membrane causing lysis or apoptosis. Likewise they are able to change the charge density of the inner membrane by the very fact that they have charge, are small and are distributed around the cell membrane from the outer surface of the membrane.
Cathelicidins contain a conserved “cathelin” precursor domain. Their organization includes an N-terminal signal peptide, a highly conserved prosequence, and a structurally variable cationic peptide at the C-terminus. The prosequence resembles cathelin, a protein originally isolated from porcine neutrophils as an inhibitor of cathepsin L (hence, the name cathelin). The 37 amino acid-long human cathelicidin, LL-37/hCAP18 has a hydrophobic N-terminal domain in an α-helical conformation, particularly in the presence of negatively charged lipids. In a step essential for its activation, LL-37 is enzymatically cleaved from the C-terminus of hCAP 18 precursor via enzymes such as neutrophil elastase and proteinase 3. LL-37 functions in synergy with other AMPs, and can directly activate host cells. Inappropriate cleavage of the cathelicidin hCAP18 pro-peptide by endogenous proteases can produce pro-inflammatory fragments of the cathelicidin (Nat Med. 2007 August; 13(8):975-80). At the same time, correct cleavage via appropriate endogenous protease processing will produce the anti-inflammatory cathelicidin analogs and peptides. Thus, a method for regulating inadequate processing of cathelicidin is required as well as a method of using the anti-inflammatory analogs or fragments to the cathelicidin peptides or pro-peptide is described and exemplified below.
The ability of cathelicidins such as LL-37 to both kill bacteria and regulate immune responses is a characteristic of numerous AMPs. The peptide can influence host immune responses via a variety of cellular interactions, for example, it has been suggest to possibly function as a chemoattractant by binding to formyl-peptide-receptor-like-1 (FPRL-1). LL-37 can recruit mast cells, and then be produced by the mast cell to kill bacteria.
AMPs exert their effects either individually or as the resultant effect of multiple AMPs. For example, in the menstrual cycle there is a monthly cycle-dependent expression of various AMPs (King A. E. et al., 2003. J. Reprod. Immunol. 59:1-16). For example, there is higher expression during the menstrual cycle of beta-defensin-2 in the menstrual stage, beta-defensin-4 in the proliferative stage, beta-defensin-3 in the early secretory stage, beta-defensin-1 in the mid secretory stage, and beta-defensin-3 in the late secretory stage. It has been suggested that maintaining the balance between the AMPs is essential for normal proliferation, differentiation and in the specific example of menstrual cycle for development. In light of the apparent roles of AMPs and most importantly of cathelicidin as was demonstrated in this and the former patent application (number WO 2004-056307) of the current inventor, cathelicidin is associated with inflammation, dysregulated cell proliferation/differentiation, dysregulated cell proliferation/differentiation balance, angiogenesis metastasis, and/or epithelial wounds, the inventor hypothesized that an optimal strategy for treating such diseases would be via methods involving decreasing the levels/activity of such AMPs/AMLs, and/or via methods involving administering such AMPs/AMLs or enhancing their expression.
The prior art approaches relating to such methods involve the previous application of the inventors in WO 2004-056307 which show that cathelicidin is an immune regulator in-vivo and therefore poses a target in treating autoimmune diseases.
The current application provides in-vivo data for specific diseases such as metabolic diseases and low grade inflammatory diseases, obesity, insulin resistance, diabetes type 2, type 1 diabetes, insulin related diabetes, osteoporosis, periodontitis, osteoarthritis, arthritic diseases, rheumatologic diseases such as rheumatoid arthritis, ankylosing spondylitis, gout and systemic lupus erythematosus, as well as multiple sclerosis, neurological and central nervous system diseases as well as osteoporosis.
In particular, the current invention shows in-vivo the use of cathelicidin or cathelicidin analogs in the treatment of said diseases.